RESUMO
Genetic polymorphisms that impair very low-density lipoprotein (VLDL) secretion are linked to hepatic steatosis, fibrosis, and hepatocellular cancer. Liver-specific deletion of microsomal triglyceride transfer protein (Mttp-LKO) impairs VLDL assembly, promoting hepatic steatosis and fibrosis, which are attenuated in Mttp-LKO X Fabp1-null [Fabp1/Mttp double knockout (DKO)] mice. The current study examined the impact of impaired VLDL secretion in Mttp-LKO mice on hepatocellular cancer incidence and progression in comparison to Fabp1/Mttp DKO mice. Diethylnitrosamine-treated Mttp-LKO mice exhibited steatosis with increased tumor burden compared with flox controls, whereas diethylnitrosamine-treated Fabp1/Mttp DKO mice exhibited a paradoxical increase in tumor burden and >50% mortality by 50 weeks. Serum high-density lipoprotein cholesterol was elevated in both Mttp-LKO and Fabp1/Mttp DKO mice, with increased intratumoral expression of apolipoprotein A1 and apolipoprotein E. Lipidomic surveys revealed progressive enrichment in distinct triglyceride species in livers from Mttp-LKO mice with further enrichment in Fabp1/Mttp DKO mice. RNA sequencing revealed mRNA changes suggesting altered monocarboxylic acid use and increased aerobic glycolysis, whereas hepatocytes from Fabp1/Mttp DKO mice exhibited increased capacity to use glucose and glutamine. These metabolic shifts were accompanied by reduced expression of HNF1 homeobox A (HNF1a), which correlated with tumor burden. Taken together, these findings demonstrate that hepatic tumorigenesis is increased in mice with impaired VLDL secretion and further accelerated via pathways including altered fatty acid compartmentalization and shifts in hepatic energy use.
Assuntos
Carcinogênese , Proteínas de Ligação a Ácido Graxo , Lipoproteínas VLDL , Neoplasias Hepáticas , Animais , Camundongos , Carcinogênese/genética , Carcinogênese/patologia , Carcinogênese/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Fígado Gorduroso/genética , Deleção de Genes , Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos KnockoutRESUMO
BACKGROUND AND AIMS: Human transmembrane 6 superfamily 2 (TM6SF2) variant rs58542926 is associated with NAFLD and HCC. However, conflicting reports in germline Tm6sf2 knockout mice suggest no change or decreased very low density lipoprotein (VLDL) secretion and either unchanged or increased hepatic steatosis, with no increased fibrosis. We generated liver-specific Tm6Sf2 knockout mice (Tm6 LKO) to study VLDL secretion and the impact on development and progression of NAFLD. APPROACH AND RESULTS: Two independent lines of Tm6 LKO mice exhibited spontaneous hepatic steatosis. Targeted lipidomic analyses showed increased triglyceride species whose distribution and abundance phenocopied findings in mice with liver-specific deletion of microsomal triglyceride transfer protein. The VLDL triglyceride secretion was reduced with small, underlipidated particles and unchanged or increased apolipoprotein B. Liver-specific adeno-associated viral, serotype 8 (AAV8) rescue using either wild-type or mutant E167K-Tm6 reduced hepatic steatosis and improved VLDL secretion. The Tm6 LKO mice fed a high milk-fat diet for 3 weeks exhibited increased steatosis and fibrosis, and those phenotypes were further exacerbated when mice were fed fibrogenic, high fat/fructose diets for 20 weeks. In two models of HCC, either neonatal mice injected with streptozotocin (NASH/STAM) and high-fat fed or with diethylnitrosamine injection plus fibrogenic diet feeding, Tm6 LKO mice exhibited increased steatosis, greater tumor burden, and increased tumor area versus Tm6 flox controls. Additionally, diethylnitrosamine-injected and fibrogenic diet-fed Tm6 LKO mice administered wild-type Tm6 or E167K-mutant Tm6 AAV8 revealed significant tumor attenuation, with tumor burden inversely correlated with Tm6 protein levels. CONCLUSIONS: Liver-specific Tm6sf2 deletion impairs VLDL secretion, promoting hepatic steatosis, fibrosis, and accelerated development of HCC, which was mitigated with AAV8- mediated rescue.
Assuntos
Carcinoma Hepatocelular/genética , Fígado Gorduroso/genética , Lipoproteínas VLDL/metabolismo , Cirrose Hepática/genética , Neoplasias Hepáticas/genética , Fígado/metabolismo , Proteínas de Membrana/genética , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Fígado Gorduroso/metabolismo , Lipidômica , Fígado/patologia , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/genética , Triglicerídeos/metabolismoRESUMO
Regulating dietary fat absorption may impact progression of nonalcoholic fatty liver disease (NAFLD). Here, we asked if inducible inhibition of chylomicron assembly, as observed in intestine-specific microsomal triglyceride (TG) transfer protein knockout mice (Mttp-IKO), could retard NAFLD progression and/or reverse established fibrosis in two dietary models. Mttp-IKO mice fed a methionine/choline-deficient (MCD) diet exhibited reduced hepatic TGs, inflammation, and fibrosis, associated with reduced oxidative stress and downstream activation of c-Jun N-terminal kinase and nuclear factor kappa B signaling pathways. However, when Mttpflox mice were fed an MCD for 5 weeks and then administered tamoxifen to induce Mttp-IKO, hepatic TG was reduced, but inflammation and fibrosis were increased after 10 days of reversal along with adaptive changes in hepatic lipogenic mRNAs. Extending the reversal time, following 5 weeks of MCD feeding to 30 days led to sustained reductions in hepatic TG, but neither inflammation nor fibrosis was decreased, and both intestinal permeability and hepatic lipogenesis were increased. In a second model, similar reductions in hepatic TG were observed when mice were fed a high-fat/high-fructose/high-cholesterol (HFFC) diet for 10 weeks, then switched to chow ± tamoxifen (HFFC â chow) or (HFFC â Mttp-IKO chow), but again neither inflammation nor fibrosis was affected. In conclusion, we found that blocking chylomicron assembly attenuates MCD-induced NAFLD progression by reducing steatosis, oxidative stress, and inflammation. In contrast, blocking chylomicron assembly in the setting of established hepatic steatosis and fibrosis caused increased intestinal permeability and compensatory shifts in hepatic lipogenesis that mitigate resolution of inflammation and fibrogenic signaling despite 50-90-fold reductions in hepatic TG.
Assuntos
Quilomícrons/metabolismo , Fígado Gorduroso/metabolismo , Fibrose/metabolismo , Inflamação/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Deficiência de Colina , Quilomícrons/antagonistas & inibidores , Dieta/efeitos adversos , Feminino , Intestinos/efeitos dos fármacos , Intestinos/metabolismo , Masculino , Metionina/deficiência , Camundongos , Camundongos Knockout , Camundongos TransgênicosRESUMO
RNA-binding proteins (RBPs) regulate diverse functions by interacting with target transcripts. Here we present a protocol to isolate RBP-mRNA complexes using RNA-CLIP and examine target mRNAs in association with ribosomal populations. We describe steps to identify specific RBPs and RNA targets reflecting a variety of developmental, physiological, and pathological states. This protocol enables RNP complex isolation from tissue sources (liver and small intestine) or populations of primary cells (hepatocytes), but not at a single-cell level. For complete details on the use and execution of this protocol, please refer to Blanc et al. (2014)1 and Blanc et al. (2021).2.
RESUMO
RNA-binding protein 47 (RBM47) is required for embryonic endoderm development, but a role in adult intestine is unknown. We studied intestine-specific Rbm47-knockout mice (Rbm47-IKO) following intestinal injury and made crosses into ApcMin/+ mice to examine alterations in intestinal proliferation, response to injury, and tumorigenesis. We also interrogated human colorectal polyps and colon carcinoma tissue. Rbm47-IKO mice exhibited increased proliferation and abnormal villus morphology and cellularity, with corresponding changes in Rbm47-IKO organoids. Rbm47-IKO mice adapted to radiation injury and were protected against chemical-induced colitis, with Rbm47-IKO intestine showing upregulation of antioxidant and Wnt signaling pathways as well as stem cell and developmental genes. Furthermore, Rbm47-IKO mice were protected against colitis-associated cancer. By contrast, aged Rbm47-IKO mice developed spontaneous polyposis, and Rbm47-IKO ApcMin/+ mice manifested an increased intestinal polyp burden. RBM47 mRNA was decreased in human colorectal cancer versus paired normal tissue, along with alternative splicing of tight junction protein 1 mRNA. Public databases revealed stage-specific reduction in RBM47 expression in colorectal cancer associated independently with decreased overall survival. These findings implicate RBM47 as a cell-intrinsic modifier of intestinal growth, inflammatory, and tumorigenic pathways.
Assuntos
Colite , Neoplasias do Colo , Adulto , Camundongos , Humanos , Animais , Idoso , Camundongos Knockout , Colite/induzido quimicamente , Colite/genética , Neoplasias do Colo/genética , Carcinogênese/genética , Proliferação de Células , RNA Mensageiro/genética , Estresse Oxidativo , Proteínas de Ligação a RNA/genéticaRESUMO
The RNA-binding protein Apobec1 complementation factor (A1CF) regulates posttranscriptional ApoB mRNA editing, but the range of RNA targets and the long-term effect of altered A1CF expression on liver function are unknown. Here we studied hepatocyte-specific A1cf-transgenic (A1cf+/Tg), A1cf+/Tg Apobec1-/-, and A1cf-/- mice fed chow or high-fat/high-fructose diets using RNA-Seq, RNA CLIP-Seq, and tissue microarrays from human hepatocellular cancer (HCC). A1cf+/Tg mice exhibited increased hepatic proliferation and steatosis, with increased lipogenic gene expression (Mogat1, Mogat2, Cidea, Cd36) associated with shifts in polysomal RNA distribution. Aged A1cf+/Tg mice developed spontaneous fibrosis, dysplasia, and HCC, and this development was accelerated on a high-fat/high-fructose diet and was independent of Apobec1. RNA-Seq revealed increased expression of mRNAs involved in oxidative stress (Gstm3, Gpx3, Cbr3), inflammatory response (Il19, Cxcl14, Tnfα, Ly6c), extracellular matrix organization (Mmp2, Col1a1, Col4a1), and proliferation (Kif20a, Mcm2, Mcm4, Mcm6), and a subset of mRNAs (including Sox4, Sox9, Cdh1) were identified in RNA CLIP-Seq. Increased A1CF expression in human HCC correlated with advanced fibrosis and with reduced survival in a subset with nonalcoholic fatty liver disease. In conclusion, we show that hepatic A1CF overexpression selectively alters polysomal distribution and mRNA expression, promoting lipogenic, proliferative, and inflammatory pathways leading to HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Fígado Gorduroso/metabolismo , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Proteínas de Ligação a RNA/genéticaRESUMO
Niemann-Pick type C1 (NPC1) disease is a fatal neurovisceral disease for which there are no FDA approved treatments, though cyclodextrin (HPßCD) slows disease progression in preclinical models and in an early phase clinical trial. Our goal was to evaluate the mechanism of action of a previously described combination-therapy, Triple Combination Formulation (TCF) - comprised of the histone deacetylase inhibitor (HDACi) vorinostat/HPßCD/PEG - shown to prolong survival in Npc1 mice. In these studies, TCF's benefit was attributed to enhanced vorinostat pharmacokinetics (PK). Here, we show that TCF reduced lipid storage, extended lifespan, and preserved neurological function in Npc1 mice. Unexpectedly, substitution of an inactive analog for vorinostat in TCF revealed similar efficacy. We demonstrate that the efficacy of TCF was attributable to enhanced HPßCD PK and independent of NPC1 protein expression. We conclude that although HDACi effectively reduce cholesterol storage in NPC1-deficient cells, HDACi are ineffective in vivo in Npc1 mice.
Assuntos
2-Hidroxipropil-beta-Ciclodextrina/uso terapêutico , Inibidores de Histona Desacetilases/uso terapêutico , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Polietilenoglicóis/uso terapêutico , Vorinostat/uso terapêutico , Animais , Células Cultivadas , Combinação de Medicamentos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/metabolismoRESUMO
The abnormal accumulation of lipids within the endolysosomal lumen occurs in many conditions, including lysosomal storage disorders, atherosclerosis, nonalcoholic fatty liver disease (NAFLD), and drug-induced phospholipidosis. Current methods cannot monitor endolysosomal lipid content in vivo, hindering preclinical drug development and research into the mechanisms linking endolysosomal lipid accumulation to disease progression. We developed a single-walled carbon nanotube-based optical reporter that noninvasively measures endolysosomal lipid accumulation via bandgap modulation of its intrinsic near-infrared emission. The reporter detected lipid accumulation in Niemann-Pick disease, atherosclerosis, and NAFLD models in vivo. By applying the reporter to the study of NAFLD, we found that elevated lipid quantities in hepatic macrophages caused by a high-fat diet persist long after reverting to a normal diet. The reporter dynamically monitored endolysosomal lipid accumulation in vivo over time scales ranging from minutes to weeks, indicating its potential to accelerate preclinical research and drug development processes.